1. Field of the Invention
This invention relates to a delivery system for introducing homogeneous oligonucleoside conjugates that are resistant to biodegradation into cells in a tissue specific manner via ligand directed, receptor mediated, endocytosis pathway.
2. Background Information
The antisense (anticode) or antigene strategy for drug design is based on the sequence-specific inhibition of protein synthesis due to the binding and masking of the target mRNA or genomic DNA, respectively, by the synthetic oligodeoxynucleotides (oligo dN) and their analogs (1). Implicit in this strategy is the ability of oligo-dns to cross cellular membranes, thereby gaining access to the cellular compartments containing their intended targets, and to do so in sufficient amounts for binding to those targets to take place. Among the many oligo-dn analogs for application as antisense, non-ionic oligonucleoside methylphosphonates (oligo-MPs) have been extensively studied (2). Oligo-MPs are totally resistant to nuclease degradation (3) and are effective antisense agents with demonstrative in vitro activity against herpes simplex virus type 1 (4), vesicular stomatitis virus (5) and human immunodeficiency virus (6), and are able to inhibit the expression of ras p21 (7). For oligo-MPs to exhibit antisense activity, however, they must be present in the extracellular medium in concentrations up to 100 .mu.M (4-7). Achieving and maintaining these concentrations for therapeutic purposes presents a number of difficulties, including expense, potential side effects owing to non-specific binding of the oligo-MP and immunogenicity. These difficulties can be circumvented in two ways:
1) By enhancing transport of the oligo-MP across the membrane of the targeted cell line achieving a locally high concentration of the oligo-MP. PA1 2) By specific delivery to a target cell line only, thereby avoiding toxic side effects to other tissues.
Both strategies serve to greatly reduce the concentration of the oligo-MP needed to produce an antisense effect and to avoid the toxic side effect with tissue specificity.
Delivery of exogenous DNA into the intracellular medium is greatly enhanced by coupling its uptake to receptor-mediated endocytosis. Pioneering work by Wu and Wu (8) showed that foreign genes (8a-c) or oligo-dns (8d), electrostatically complexed to poly-L-lysine linked to asialoorosomucoid, are efficiently and specifically taken into human hepatocellular carcinoma (Hep G2) cells through direct interaction with the asialoglycoprotein receptor. Since this initial study, other examples of receptor-mediated delivery of DNA have appeared including a tetra-antennary galactose neoglycopeptide-poly-L-lysine conjugate (9), a trigalactosylated bisacridine conjugate (10), folate conjugates (11), an antibody conjugate (12), transferrin conjugate (13) and 6-phosphomannosylated protein linked to an antisense oligo-dns via a disulfide bond (14). Recently, the tri-antennary N-acetylgalactosamine neoglycopeptide, YEE(ah-GalNAc).sub.3 (15), conjugated to human serum albumin which was in turn linked to poly-L-lysine was shown to effectively deliver DNA into Hep G2 cells (16). While improved, these methods of delivery have several disadvantages: (1) by virtue of the structural heterogeneity of the starting materials (e.g. most often poly-L-lysine or bovine serum albumin) and the synthetic strategies employed, glycoconjugates derived from these materials are functionally equivalent, but structurally heterogeneous, therefore, their physical and biological properties would be difficult to fully define; (2) polycationic compounds (e.g. poly-L-lysine and cationic lipids) are toxic at concentrations employed for the delivery of DNA and oligo-dns in vitro and presumably in vivo; (3) the ratio of oligo-dN or DNA to cationic conjugate must be empirically determined in each case.
A number of synthesis products have been described for the delivery of oligo dN which are heterogeneous mixtures of conjugates. Bonfils et al., for example, describe formation of conjugates between 6-phosphomannosylated protein and oligonucleosides which, because the modification of the protein and the formation of the disulfide link are not regiochemically controlled, yields a mixture of functionally related but structurally different molecules.
Several studies have described intracellular delivery of oligodeoxynucleotides or DNA which contain biodegradable phosphodiester internucleotide linkages. Because of this, they may have relatively short half lives within the cell and efficacy is consequently reduced. For example, an all phosphodiester 16-mer, d(T).sub.16, was extensively degraded after only two hours in the cell. This disadvantage with oligo-dNs and DNA is well recognized in the antisense community. The present invention enables the delivery of biologically stable oligo-MPs, other oligodeoxynucleotide analogs (see Table 1) and other biostable pro-drugs using the delivery strategy described herein. Thus, a further reduction in the concentration of the antisense agent can be realized as a consequence of its biostability.
Merwin et al. describe the synthesis of conjugates using neoglycopeptide YEE(ah-GalNAc).sub.3. Their delivery system is heterogeneous and contains poly-L-lysine, which serves to electrostatically bind DNA to the conjugate. The disadvantages of this delivery strategy are: its structural heterogeneity; potential toxicity due to its polycationic charge; and difficulties in formulation due to the need to empirically determine the ratio of cationic carrier to oligo-dn or DNA for optimum delivery. The delivery strategy of the present invention obviates all these difficulties because it is chemically defined and structurally homogeneous and it is intended to deliver biologically stable antisense agents and other biostable pro-drugs.
Thus, conjugates available heretofor for delivering oligo dN and oligo dN analogs to their intracellular targets suffer from the disadvantage of being heterogeneous and/or rapidly biodegradable, with the consequences that the most efficacious compounds may be delivered in a dilute form to the target along with extraneous compounds which may be ineffective and/or harmful.